Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide secreted by the nerves of the central and peripheral nervous systems, where CGRP-containing neurons are closely associated with blood vessels. CGRP-mediated vasodilatation is also associated with neurogenic inflammation, as part of a cascade of events that results in extravasation of plasma and vasodilatation of the microvasculature and is present in migraines.
CGRP exists as highly homologous α and β isoforms in both human and rat although each is encoded by a distinct gene. The α and β isoforms of the CGRP peptides differ by three amino acids in humans and one amino acid in rats. The amino acid sequences of CGRP peptides are well conserved among species and are considered members of a family of peptides that includes amylin, calcitonin, and adrenomedullin.
CGRP is thus divided into at least two subtypes, denoted as α-CGRP or CGRP1 and β-CGRP or CGRP2 (Pharmacol. Rev 54:233-246, 2002). The existence of at least two CGRP subtypes had been proposed from differential antagonist affinities and agonist potencies in a variety of in vivo and in vitro bioassays. (Dennis et al., hCGRP8-37, A calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery, J. Pharmacol. Exp. Ther., 254:123-128 (1990); Dennis et al., Structure-activity profile of calcitonin gene-related peptide in peripheral and brain tissues. Evidence for receptor multiplicity, J. Pharmacol. Exp. Ther., 251:718-725 (1989); Dumont et al., A potent and selective CGRP agonist, [Cys(Et)2,7]hCGRP alpha: comparison in prototypical CGRP1 and CGRP2 in vitro bioassays, Can. J. Physiol. Pharmacol, 75:671-676 (1997)).
The CGRP1 subtype was found to be sensitive to the antagonist fragment CGRP(8-37). (Chiba et al., Calcitonin gene-related peptide receptor antagonist human CGRP-(8-37), Am. J. Physiol, 256:E331-E335 (1989); Dennis et al (1990); Mimeault et al, Comparative affinities and antagonistic potencies of various human calcitonin gene-related peptide fragments on calcitonin gene-related peptide receptors in brain and periphery, J. Pharmacol. Exp. Ther., 258:1084-1090 (1991)). By contrast, the CGRP was sensitive to linear human CGRP (hCGRP) analogs, in which the cysteine residues at positions 2 and 7 were derivatized (e.g., with acetoaminomethyl [Cys(ACM)2′7] or ethylamide [Cys(Et)2′7]) but CGRP receptor was insensitive to fragment CGRP(8-37). (Dennis et al (1989); Dennis et al 1990); Dumont et al (1997)). Three calcitonin receptor stimulating peptides (CRSPs) have also been identified in a number of mammalian species; the CRSPs may form a new subfamily in the CGRP family. (Katafuchi, T and Minamino, N, Structure and biological properties of three calcitonin receptor-stimulating peptides, novel members of the calcitonin gene-related peptide family, Peptides, 25(11):2039-2045 (2004)).
CGRP mediates its effects through a heteromeric receptor composed of a G protein-coupled receptor called calcitonin receptor-like receptor (CALCRL) and a receptor activity-modifying protein (RAMP1). CGRP receptors have been identified and pharmacologically evaluated in several tissues and cells, including brain, cardiovascular, endothelial and smooth muscle. Multiple CGRP receptors have been characterized based on distinct pharmacological properties. The calcitonin superfamily peptides act through seven-transmembrane-domain G-protein-coupled receptors (GPCRs). The calcitonin receptor (“CT”, “CTR” or “CT receptor”) and CGRP receptors are type II (“family B”) GPCRs, which family includes other GPCRs that recognize regulatory peptides such as secretin, glucagon and vasoactive intestinal polypeptide (VIP). The best characterized splice variants of human calcitonin receptor differ depending on the presence (formerly CTRn+ or CTRI, now known as CT{circumflex over ( )})) or absence (the major splice variant, formerly CTRπ_ or CTR2, now known as CT(a)) of 16 amino acids in the first intracellular loop. (Gorn et al., Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone: the first intracellular domain modulates ligand binding and signal transduction, J. Clin. Invest., 95:2680-2691 (1995); Hay et al., Amylin receptors: molecular composition and pharmacology, Biochem. Soc. Trans., 32:865-867 (2004); Poyner et al., 2002).
CGRP is widely distributed in sensory nerves, both in the peripheral and central nervous system and displays a large number of different biological activities. When released from trigeminal and other nerve fibers, CGRP is thought to mediate its biological responses by binding to specific cell surface receptors. The biological activities of CGRP include the regulation of neuromuscular junctions, of antigen presentation within the immune system, of vascular tone and of sensory neurotransmission. (Poyner, D. R., Calcitonin gene-related peptide: multiple actions, multiple receptors, Pharmacol. Ther., 56:23-51 (1992); Muff et al., Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions, Eur. J. Endocrinol., 133: 17-20 (1995)).
There is a great need, therefore, to identify new compounds that specifically recognize and bind CGRP. Such compounds would be useful for diagnostic screening and therapeutic intervention in disease states that are associated with CGRP activity. Accordingly, it is an object of the present disclosure to provide specific binding compounds of CGRP for modulating CGRP activity. Such compounds disclosed herein take the form of muteins derived from human lipocalin 2 (also known as neutrophil gelatinase associated lipocalin, “hNGAL”).